Butanol or butyl alcohol (sometimes also called biobutanol when produced biologically), is a primary alcohol with a 4 carbon structure and the molecular formula of C4H10O. It is primarily used as a solvent, as an intermediate in chemical synthesis, and as a fuel. Today, there is a paramount interest in producing fuels like butanol and ethanol using microorganisms by fermentation focusing on the environmental aspects and renewable nature of this mode of production. Butanol is a superior, fuel and has more calorific value than ethanol (Qureshi and Blaschek, 2000). Butanol has higher energy content than ethanol (110,000 Btu's per gallon for butanol vs. 84,000 Btu per gallon for ethanol). It is six times less “evaporative” than ethanol and 13.5 times less evaporative than gasoline, can be shipped through existing fuel pipelines where ethanol must be transported via rail, barge or truck (Jones and Woods, 1986).
Butanol is an important industrial chemical. Compared to the currently popular fuel additive ethanol, butanol is more miscible with gasoline and diesel fuel, has a lower vapor pressure, and is less miscible with water, qualities that make butanol a superior fuel extender than ethanol. Current butanol prices as a chemical are at $3.75 per gallon, with a worldwide market of 370 million gallons per year. The market demand is expected to increase dramatically if green butanol can be produced economically from low cost biomass. In addition to its usage as fuel, butanol can be used as a solvent for a wide variety of chemical and textile processes, in organic synthesis and as a chemical intermediate. It is also used as paint thinner and a solvent in other coating applications where it is used as a relatively slow evaporating latent solvent in lacquers and ambient-cured enamels. It finds other uses such as a component of hydraulic and brake fluids (Mutschlechner et al., 2000). It is also used as a base for perfumes, but on its own has a highly alcoholic aroma.
Since the 1950s, most butanol in the United States is produced commercially from fossil fuels. The most common process starts with propene, which is run through an hydroformylation reaction to form butanal, which is then reduced with hydrogen to butanol. Butanol is produced by fermentation, from corn, grass, leaves, agricultural waste and other biomass.
Production of industrial butanol and, acetone via fermentation, using Clostridium acetobutylicum, started in 1916. Chaim Weizmann, a student of Louis Pasteur, isolated the microbe that made acetone. Up until the 1920s, acetone was the product sought, but for every pound of acetone fermented, two pounds of butanol were formed. A growing automotive paint industry turned the market around, and by 1927 butanol was primary and acetone became the byproduct.
The production of butanol by fermentation declined from the 1940s through the 1950s, mainly because the price of petrochemicals dropped below that of starch and sugar substrates such as corn and molasses. The labor intensive batch fermentation system's overhead combined with the low yields contributed to the situation. Fermentation-derived acetone and butanol production ceased in the late 1950s.
Acetone butanol ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations. It was ranked second only to ethanol fermentation by yeast in its scale of production, and is one of the largest biotechnological processes ever known. The actual fermentation, however, has been quite complicated and difficult to control. ABE fermentation has declined continuously since the 1950s, and almost all butanol is now produced via petrochemical routes. In a typical ABE fermentation, butyric, propionic, lactic and acetic acids are first produced by C. acetobutylicum, the culture pH drops and undergoes a metabolic “butterfly” shift, and butanol, acetone, isopropanol and ethanol are formed. In conventional ABE fermentations, the butanol yield from glucose is low, typically around 15 percent and rarely exceeding 25 percent.
The production of butanol was limited by severe product inhibition. Butanol at a concentration of 1 percent can significantly inhibit cell growth and the fermentation process. Consequently, butanol concentration in conventional ABE fermentations is usually lower than 1.3 percent. The key problem associated with butanol production is butanol toxicity/inhibition of the fermenting microorganism, resulting in low butanol titer in the fermentation broth. (Ezeji et al., 2007). Butanol is highly toxic to biological systems at quite low concentrations of 2% (Jones and Wood, 1986). This toxicity may be because butanol localizes in the plasma membrane and disrupts a number of physiological processes including membrane permeability, solute transport, maintenance of proton motive force, conformation and activity of intrinsic membrane proteins. Efforts are being made to improve the butanol tolerance level in different species of Clostridia with varying degree of success (Evan and Wang, 1988). Recent interest in the production of butanol has lead to re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture.
In the past 20+ years, there have been numerous engineering attempts to improve butanol production in ABE fermentation, including cell recycling and cell immobilization to increase cell density and reactor productivity and using extractive fermentation to minimize product inhibition. Despite many efforts, the best results ever obtained for ABE fermentations to date are still less than 2 percent in butanol concentration, 4.46 g/L/h productivity, and a yield of less than 25 percent from glucose. Optimizing the ABE fermentation process has long been a goal of the industry.
With that in mind, an alternative process was developed using continuous immobilized cultures of Clostridium tyrobutyricum and Clostridium acetobutylicum to produce an optimal butanol productivity of 4.64 g/L/h and yield of 42 percent. In simple terms, one microbe maximizes the production of hydrogen and butyric acid, while the other converts butyric acid to butanol. Compared to conventional ABE fermentation, this process eliminates acetic, lactic and propionic acids, acetone, isopropanol and ethanol production. The ABE fermentation process only produces hydrogen, butyric acid, butanol and carbon dioxide, and doubles the yield of butanol from a bushel of corn from 1.3 to 2.5 gallons per bushel. The drawbacks associated with such a process are two folds: having to maintain two sets of conditions for the two cultures, maintaining complete anaerobiosis in the immobilized system, dealing with the gases produced during the fermentation and their effect on the maintaining the integrity of the matrix used for immobilization.
In conventional ABE fermentations, the butanol yield, from glucose is low—between 15%-25%—and the butanol concentration in the fermentation is usually lower than 1.3%. (Butanol at a concentration of 1% can significantly inhibit cell growth and the fermentation process.). There have been numerous efforts over the years to improve butanol yield by using a variety of techniques to minimize product inhibition.
In this respect, to develop a process for the maximum production and tolerance of this important fuel by process designing, standardization of media and fermentation conditions, strain improvement is of utmost importance (Agarwal et al., 2005). Physiological and nutritional factors such as initial sugar concentration, complex nitrogen sources, inoculum size, carbonate ion concentrations, pH and temperature of the growth medium are reported to be the most critical factors affecting both cell growth and butanol production (Samuelov et al., 1991; Nghiem et al., 1997; Lee et al., 1999).
U.S. Pat. No. 4,757,010 and European patent application EP 00111683 provides an improved strain of Clostridium for increased tolerance to butanol. JP03058782 provides Clostridium pasteurianum CA 101 stock (FERM P-10817) as a mutant of genus Clostridium bacterium having analog resistance to fermented intermediate of butanol and butanol producibility. U.S. Pat. No. 4,539,293 demonstrates the use of co-culture of microorganisms of the Clostridium genus, one favors the production of butyric acid and the other supports the formation of butanol. Japanese patent application JP 63157989 provides production of butanol by culturing a different strain Clostridium pasteurianum var. 1-53 (FERM P-9074) in a liquid medium containing a carbon source, a nitrogen source and other inorganic salts at 28-33° C. under slightly acidic pH condition in anaerobic state for 2-4 days.
However the problems associated in these modified strains is that the use of genetically modified strains for fuel production cannot compete with the wild type as one needs to sterilize the feedstock to make sure that there is no competition for the genetically modified organisms. Further genetically modified organisms or various strains are expensive to develop and does not find relevance on high volume products.
Various alternative in situ/online techniques of butanol removal including membrane-based systems such as pervaporation, liquid-liquid extraction, and gas stripping are used.
U.S. Pat. No. 4,777,135 describes a method of producing butanol by fermentation which comprises culturing under anaerobic conditions a butanol-producing microorganisms in a culture medium containing fluorocarbons. This process is not feasible on a commercial scale as the fluorocarbons are environmentally not safe
U.S. Pat. No. 4,605,620 provides a process for butanol by using a medium containing carbohydrate and phosphate, wherein the experiments were performed with a total phosphate content of 1.0-0.4 mmoles. This process poses a restriction wherein the phosphate limiting medium is required.
U.S. Pat. No. 4,560,658 provides a process for the production of butanol by fermentation of carbon containing compounds with Clostridium acetobutylicum wherein the fermentation is conducted in an aqueous medium containing a sufficient concentration of dissolved carbon monoxide. However the use of carbon monoxide make the process environmentally unsound.
U.S. Pat. No. 4,520,104 provides a process for the continuous production of butanol by fermentation of carbohydrates with C. acetobutylicum. This process combines continuous inoculum production at a high dilution rate and cycling the fermentation broth through material which adsorbs butanol whereby a vigorous cell population is maintained in the fermentation reactor for extended periods of time. The process is devised to remove the butanol produced in the broth so as to prevent its toxicity on the cells
Japanese patent JP 62278989 provides a fermentation process for the production of acetone and butanol, by keeping a butanol-producing strain in resting state, adding a carbon source to the cell to effect the production of acetone and butanol in a short time, recovering and concentrating the butanol-producing strain, subjecting to the heat shock and adding to a fermentation tank Heat shock is required in the process.—to activate the spores of Clostridium and is pretty routine.
Japanese patent application provides an anaerobic cellulolytic germ, e.g. Clostridium cellobioparum ATCC15832 or Ruminococcus albus ATCC27211, and Clostridium saccharoperbutylacetonicum are inoculated into a culture medium containing a material containing cellulose, e.g. wood, waste paper or pulp, as a main carbon source, and cultivated at 25-45° C. and 4-9 pH under anaerobic conditions for about 2-20 days to collect the aimed compound, containing oxygen, and consisting essentially of butanol from the resultant culture. This process is time consuming and takes about 20 days for completion, hence not feasible on a large scale.
Japanese patent 63269988 discloses butanol fermentation wherein yeast is subjected to autodigestion in a fermentation tank and proliferated prior to the inoculation of butanol-producing strain. The space in the fermentation tank becomes anaerobic and the temperature increases by the proliferation of yeast to perform butanol fermentation. An inefficient autodigestion would lead to contamination of the broth by the yeast
US20050233031 provides a process for producing butanol which includes treating plant derived material to provide an aqueous liquor containing sugars in a fermentation process to produce a fermentation product. The process involves several steps and therefore cumbersome and tedious.
Japanese Patent JP 200535328801 provides a method for producing butanol in which a culture solution is prepared by using a formulation of the food residue with the Japanese distilled spirit lees and water and butanol fermentation is carried out in the culture solution. The use of Japanese distilled spirit is limited to the production experiments performed in Japan.
French patent FR2550222 provides a two stage process wherein a first stage of seeding with Clostridium acetobutylicum and a second stage of seeding with a yeast which produces ethanol, the second stage being commenced when the pH of the fermentation medium of the first stage has reached a minimum value. The invention applies in particular to the production of butanol, acetone and ethanol from sugarbeet and Jerusalem artichoke juices. The process requires pretreatment which makes it cumbersome.
Although, there are reports where microbes have been exploited for the production of butanol by fermentation, an economically viable biosynthetic process for butanol production is yet to be developed (Jesse et al., 2002).
Mustafa et al provided mid-infrared spectroscopy coupled to sequential injection analysis for the on-line monitoring of the acetone—butanol fermentation process. This involves the use of highly sophisticated instruments/skills which are not available in many laboratories. (Mustafa K. et al., Spectroscopy Letters, 38, 677-702 (2005))
Gas chromatography and gateway sensors for on-line state estimation of complex fermentations (butanol-acetone fermentation) showed a fermentation system that has been designed to demonstrate the use of gas chromatography (GC) for on-line monitoring of the butanol-acetone and other complex saccharolytic fermentations. (McLaughlin J K, Meyer C L, Papoutsakis E T. (1985) Biotechnology and Bioengineering Volume 27, Issue 8, Pages 1246-1257). However, parameters include glucose concentration and gas composition, as well as a number of unobservable parameters (such as YATP, excess ATP, and NAD reduced by FdH2), which characterize the state of the fermentation. Hence this method is very tedious requiring numerous parameters to be monitored.
U.S. Pat. Nos. 4,521,516, 4,520,104, 4,560,658, 4,649,112 disclose methods of HPLC determination of butanol wherein the components were analyzed chromatographically by elution with 0.006N H2SO4 from a cation-exchange resin in the hydrogen form. The eluted components were detected by means of a differential refractometer, plotted on a recorder and quantitated using an electronic integrator. The area under the curve which represents the concentration of each component is reported as a percentage of the total area. The general procedure followed was that given in “Analysis of Carbohydrate Mixtures by Liquid Chromatography”, Am. Soc. Brew. Chem. Proc., 1973, pp. 43-46. The separations were made on a 1-foot HPX-87 column in the hydrogen form, available from Bio-Rad Laboratories, Richmond, Calif. The Residual total carbohydrate (RTC) in the fermentation medium was measured by the phenol/sulfuric acid method which has been described in detail by Dubois, et al, “Colorimetric Method Determination of Sugars and Related Substances”, Anal. Chem., 28, 350-356 (1956)
Hence these above cited methods where generally GC is used for the estimation of butanol (Bryant and Blaschek, 1988). requires extraction or derivatization of butanol in hexane or other solvents before analyzing the samples. This makes the process tedious and there may be some losses during the extraction or derivatization steps. A couple of HPLC methods have been reported (Ehrlich et al., 1981) for the estimation of butanol. But even in these methods, the run time is too long (30-50 min) to detect the butanol. Hence such a method can not be used for analyzing large number of samples.